Understanding and manipulating hematopoietic differentiation requires knowing the regulatory circuitry that orchestrates the programs of gene expression during this process. One class of gene regulatory molecules are the microRNAs (miRNAs) - tiny endogenous RNAs, about 22 nucleotides in length, that are thought to use the elements of the RNA-interference pathway to post transcriptionally down-regulate the expression of protein-coding genes. Starting with the hypothesis that miRNAs are playing important regulatory roles during hematopoietic differentiation, the Lodish and Bartel labs have collaborated to clone about 100 different miRNAs from mouse bone marrow. These include five miRNAs referred to as "hematopoietic miRNAs", because they are highly or preferentially expressed in hematopoietic cell lineages. Three of the five also derive from loci associated with chromosomal breakpoints or aberrations previously linked to leukemias. Preliminary studies show that ectopic expression of one of these miRNAs in bone marrow progenitors modulates hematopoietic differentiation both in cell culture and in transplanted mice. The experiments of this proposal focus on the hematopoietic miRNAs with the broad, long-term objective of understanding the gene regulatory events needed for hematopoietic stem cell and progenitor maintenance and differentiation. The specific aims are: 1) To examine the consequences of altered miRNA expression during hematopoiesis. 2) To identify the regulatory targets of hematopoietic miRNAs and examine the consequences of disrupting miRNA regulation of these targets. 3) To identify additional hematopoietic miRNAs. These experiments include the ectopic expression of hematopoietic miRNAs in hematopoietic stem cells and lineage-committed progenitors, knock-outs of miRNA genes, in vitro validation of predicted miRNA regulatory targets, in vivo substitution of target genes with versions unresponsive to miRNA regulation, cloning of additional miRNAs from hematopoietic tissues, and further expression analyses. They seek to place miRNAs within specific gene regulatory pathways needed for hematopoietic stem cell maintenance and lymphoid and myeloid differentiation. They will also address more fundamental issues regarding miRNA regulation, such as the combinatorial control of expression by miRNAs and the features of functional miRNA complementary sites within vertebrate mRNAs. Thus, these experiments will provide important insights for understanding the action of miRNAs in mammals and how their dysfunction might contribute to both hematological and other human diseases.